Peptides, in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, agrochemical or cosmetic ingredient.
It is known that oligopeptides can be chemically synthesized in solution or on the solid phase via highly optimized processes. However, there are still some limitations in chemical peptide synthesis especially on large scale. For instance, peptides longer than 10-15 amino acids are difficult to synthesize on the solid phase because they tend to form tertiary structures (by so-called “hydrophobic collapse”) making peptide elongation very troublesome so that a large excess of reagents and amino acid building blocks is needed. Additionally the purification of the final product is often cost-inefficient due to the presence of significant amounts of peptides of similar length. Therefore, peptides longer than 10 amino acids are often synthesized by a combination of solid phase synthesis of protected oligopeptide fragments which are subsequently chemically condensed in solution, e.g. a 10+10 condensation to make a peptide of 20 amino acids. The major drawback of chemical protected oligopeptide fragment condensation is that upon activation of the C-terminal amino acid residue racemisation occurs, except when C-terminal Gly or Pro residues are used. Therefore, the chemical protected oligopeptide fragment condensation strategy is limited to using C-terminally activated Gly and Pro residues, or one has to deal with a very difficult purification due to the formation of undesired diastereoisomers. In contrast, enzyme-catalyzed oligopeptide couplings are completely devoid of racemisation and have several other advantages over chemical peptide synthesis. For industrial application, an enzymatic peptide synthesis concept based on a kinetic approach, i.e. using an activated carboxy component is most attractive (see for instance Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology”, 1st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002).
Chemo-enzymatic peptide synthesis can entail the enzymatic coupling of side-chain unprotected oligopeptide fragments which have individually been synthesized using chemical synthesis, fermentation, or by a combination of chemical and enzymatic coupling steps. Some reports have been published on the enzymatic condensation of fully side-chain unprotected oligopeptides in aqueous environment (Kumaran et al. Protein Science, 2000, 9, 734; Bjorup et al. Bioorg. Med. Chem. 1998, 6, 891; Homandberg et al. Biochemistry, 1981, 21, 3387; Komoriya et al. Int. J. Pep. Prot. Res. 1980, 16, 433). However, a major drawback of such enzymatic oligopeptide fragment condensation in aqueous systems is that simultaneous hydrolysis of the oligopeptide amide bonds and of the C-terminal ester takes place leading to low yields and many side products. To lower the amount of hydrolysis of the expensive oligopeptide starting materials and peptide products, often a large excess of oligopeptide nucleophile is used (5-10 equivalents) to increase the condensation rate and hence decrease the hydrolytic side reactions, which is economically a very unattractive strategy. To further lower the amount of hydrolysis, enzymatic fully unprotected oligopeptide fragment condensations have been performed in low-aqueous reaction mixtures using organic co-solvents showing higher product yields and less hydrolytic side reactions (Slomczynska et al. Biopolymers, 1992, 32, 1461; Xaus et al. Biotechnol. Tech. 1992, 6, 69; Nishino et al. Tet. Lett. 1992, 33, 3137; Clápes et al. Bioorg. Med. Chem. 1995, 3, 245, Kolobanova et al. Russian J. of Bioorg. Chemistry 2000, 26, 6, 369). Because in these reports a significant amount of water is required for enzyme activity (between 1-5 vol % of water), hydrolytic side reactions are still not fully eliminated. To virtually eliminate enzymatic hydrolytic side reactions, near anhydrous reaction mixtures can be used (below 1 vol % water). However there are only very few enzymes active and stable under these conditions (G. Carrea, S. Riva, Fundamentals of Biocatalysis in Neat Organic Solvents, Whiley, 2008) and oligopeptides containing unprotected side-chain functionalities usually display very little or no solubility in these organic solvents. Some reports have been disclosed on the enzymatic synthesis of di- and tri-peptides in anhydrous organic solvents (e.g. Chen et al. J. Org. Chem. 1992, 57, 6960), but no oligopeptide fragment condensations have been performed. Although near anhydrous solvents virtually eliminate hydrolytic side reactions, most often much enzyme activity is lost and thus oligopeptide coupling reactions tend to be very slow and incomplete.
As is known from solution phase chemical peptide synthesis, protected oligopeptides are well soluble in several neat organic solvents due to their hydrophobic character. Thus, enzymatic oligopeptide fragment condensation in anhydrous organic solvents might be performed using protected oligopeptides. However, one would expect that multiple sterically demanding hydrophobic side-chain protecting groups block enzyme recognition. For instance, it was reported by Yan et al. Tetrahedron, 2005, 61, 5933 that no condensation products were obtained at all with side-chain protected amino acids using the protease subtilisin A. According to their observations subtilisin A does not except amino acid residues bearing bulky protecting groups on their side chain functionality; however, when these bulky protecting groups are removed, the amino acid residues are readily accepted.
Gill et al (J. Am. Chem. Soc 1995, 117, 6175-6181) also describe a method for the enzymatic synthesis of oligopeptides. However, the synthesis as described by Gill et al requires specific enzymes for every individual addition of amino acids to synthesize fragments, and another enzyme again for the coupling of two fragments. The fact that different enzymes have to be used for the assembly of the two fragments and another enzyme for the condensation of the two fragments, makes the application of this process on an industrial scale unattractive. Moreover, the fragment condensation step as described by Gill et al requires the removal of two side chain allyl protecting groups of one of the fragments, to allow the use of V8 protease to achieve the coupling. Thus, the process as described by Gill et al is not a versatile process for the preparation of oligopeptides comprising 8 amino acid residues or more.